Abstract
Autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS) is a multisystem hereditary ataxia associated with mutations in SACS, which encodes sacsin, a protein of still only partially understood function. Although mouse models of ARSACS mimic largely the disease progression seen in humans, their use in the validation of effective therapies has not yet been proposed. Recently, the teleost Danio rerio has attracted increasing attention as a vertebrate model that allows rapid and economical screening, of candidate molecules, and thus combines the advantages of whole-organism phenotypic assays and in vitro high-throughput screening assays. Through CRISPR/Cas9-based mutagenesis, we generated and characterized a zebrafish sacs-null mutant line that replicates the main features of ARSACS. The sacs-null fish showed motor impairment, hindbrain atrophy, mitochondrial dysfunction, and reactive oxygen species accumulation. As proof of principle for using these mutant fish in high-throughput screening studies, we showed that both acetyl-DL-leucine and tauroursodeoxycholic acid improved locomotor and biochemical phenotypes in sacs−/− larvae treated with these neuroprotective agents, by mediating significant rescue of the molecular functions altered by sacsin loss. Taken together, the evidence here reported shows the zebrafish to be a valuable model organism for the identification of novel molecular mechanisms and for efficient and rapid in vivo optimization and screening of potential therapeutic compounds. These findings may pave the way for new interventions targeting the earliest phases of Purkinje cell degeneration in ARSACS.
Highlights
Autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS) is a rare earlyonset neurodegenerative disease associated with mutations in the SACS gene, which encodes sacsin, a 520 kDa multidomain protein [1]
From the N- to the C-terminal, sacsin is reported to be composed of a ubiquitin-like domain that binds to the proteasome, three large sacsin repeats forming a “sacsin repeating region” (SRR) that may have an Hsp90-like chaperone function [5], a xeroderma pigmentosum complementation group C binding (XPCB) domain that binds to the Ube3A ubiquitin protein ligase, a DnaJ domain binding Hsc70, and a higher eukaryotes and prokaryotes nucleotide-binding (HEPN) domain mediating sacsin dimerization and binding to nucleotides or their analogs [1,5,6]
Even more recently, using an original proteomic approach in neuronal-like cells, we identified significant dysregulation of biological processes related to neuroinflammation, synaptogenesis, and engulfment of cells in ARSACS, these findings further reinforcing the hypothesis of a role for sacsin in neurodevelopment [13]
Summary
Autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS) is a rare earlyonset neurodegenerative disease associated with mutations in the SACS gene, which encodes sacsin, a 520 kDa multidomain protein [1]. The SRR has recently been found to be part of a larger sacsin internal repeat (SIRPT) region [7] The nature of these modules and their architecture suggest that sacsin may be involved in protein quality control, and play a role in neurodevelopment and neurodegeneration. ARSACS remains an incurable disorder and there is an urgent need to define new therapies In this context, high-capacity in vivo screening of candidate drugs/compounds would be useful both for optimizing compounds and for prioritizing their subsequent testing in mammalian models. With the dual aim of increasing our firepower in the battle against ARSACS and better characterizing biochemical alterations driven by the absence of sacsin, we developed a new loss-of-function vertebrate model in order to shed further light on the role of sacsin in early neurodevelopment, and evaluate the efficacy of the system in drug screening. Sci. 2021, 22, 8401 for the translation of potential therapies to ARSACS patients, as it decreased the need for time-consuming and labor-intensive procedures
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